Glucagon is thought to regulate hepatic carbohydrate metabolism via the classical cyclic AMP dependent mechanism, while it now appears that catecholamines act through a Ca 2 ion sensitive, alpha-adrenergic mechanism that does not involve cyclic AMP dependent protein kinase. However, the applicant's recent work has shown that both pathways control metabolism by increasing the phosphorylation of regulatory enzymes such as phosphorylase, glycogen synthase and pyruvate kinase. The objective of this proposal is to define the biochemical reactions responsible for the increase in enzyme phosphorylation following stimulation of hepatocytes via the alpha-adrenergic pathway. This goal will be met by a combination of three approaches. One approach will be to identify the substrates phosphorylated via the cyclic AMP independent pathway following stimulation of the intact cell. The phosphorylated enzymes will be separated and identified using high resolution, two dimensional gel electrophoresis. A second approach will be to explore the differences in the molecular properties of phosphorylase, glycogen synthase and pyruvate kinase following stimulation of their phosphorylation in the intact cell with both glucagon and catecholamines. The kinetics of each enzyme and the site phosphorylated in the molecule will be compared following stimulation through both pathways. The third approach will be to purify Ca2 ions sensitive, cyclic AMP independent kinases from liver and incubate them with phosphorylase, glycogen synthase and pyruvate kinase in an attempt to reproduce the effects observed in the intact cell. The knowledge gained in the first two approaches will be used as a guide toward selection of the relevant kinase.